steroids 73 (2008) 1160–1173

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Heterologous expression of human mPR␣, mPR␤ and mPR␥ in yeast confirms their ability to function as membrane receptors

Jessica L. Smith, Brian R. Kupchak, Ibon Garitaonandia 1, L. Kim Hoang, Andrew S. Maina, Lisa M. Regalla 2, Thomas J. Lyons ∗

Department of Chemistry, University of Florida, P.O. Box 117200, Gainesville, FL 32611, United States article info abstract

Article history: The nuclear progesterone (nPR) mediates many of the physiological effects of Received 1 April 2008 progesterone by regulating the expression of , however, progesterone also exerts Received in revised form 6 May 2008 non-transcriptional (non-genomic) effects that have been proposed to rely on a receptor Accepted 8 May 2008 that is distinct from nPR. Several members of the progestin and AdipoQ-Receptor (PAQR) Published on line 18 May 2008 family were recently identified as potential mediators of these non-genomic effects. Mem- branes from cells expressing these , called mPR␣, mPR␤ and mPR␥, were shown to Keywords: specifically bind progesterone and have G- coupled receptor (GPCR) characteristics, Membrane progestin receptors although other studies dispute these findings. To clarify the role of these mPRs in non- Progesterone genomic progesterone signaling, we established an assay for PAQR functional evaluation Yeast using heterologous expression in Saccharomyces cerevisiae. Using this assay, we demonstrate ␣ ␤ ␥ Adiponectin unequivocally that mPR , mPR and mPR can sense and respond to progesterone with EC50 Osmotin values that are physiologically relevant. Agonist profiles also show that mPR␣, mPR␤ and mPR␥ are activated by ligands, such as 17␣-hydroxyprogesterone, that are known to activate non-genomic pathways but not nPR. These results strongly suggest that these receptors may indeed function as the long-sought-after membrane progesterone receptors. Additionally, we show that two uncharacterized PAQRs, PAQR6 and PAQR9, are also capable of respond- ing to progesterone. These mPR-like PAQRs have been renamed as mPR␦ (PAQR6) and mPR␧ (PAQR9). Additional characterization of mPR␥ and mPR␣ indicates that their progesterone- dependent signaling in yeast does not require heterotrimeric G-proteins, thus calling into question the characterization of the mPRs as a novel class of G-protein coupled receptor. © 2008 Elsevier Inc. All rights reserved.

1. Introduction This type of signal transduction is often referred to as genomic signaling because this receptor, also called the nuclear proges- The classic paradigm for progesterone-mediated signal trans- terone receptor (nPR), doubles as a DNA binding transcription duction involves diffusion of the steroid hormone into cells factor and, as such, modulates transcription in response and binding to soluble intracellular progesterone receptors [1]. to progesterone. It has long been recognized, however, that

∗ Corresponding author. Tel.: +1 352 846 3392; fax: +1 352 846 2095. E-mail address: [email protected]fl.edu (T.J. Lyons). 1 Current address: Department of Neurobiology, SBR-14, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, United States. 2 Current address: Boston Museum of Science, Science Park, Boston, MA 02114, United States. 0039-128X/$ – see front matter © 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.steroids.2008.05.003 steroids 73 (2008) 1160–1173 1161

many of the physiological effects of progesterone occur far too attribute progesterone-dependent effects solely to one pro- rapidly to require gene transcription and often occur in cells tein. This line of research is beyond the scope of this study that are transcriptionally silent, such as sperm [2]. Moreover, and, ultimately, the discovery of the true physiological roles the receptor(s) responsible for mediating the non-genomic of mPR␣,mPR␤ and mPR␥ may require the development of effects of progesterone have a markedly different agonist pro- mouse knockout strains. files from nPR [3], suggesting that it is a distinct receptor. Herein, we address another important aspect of character- These facts have led researchers to propose the existence of izing mPR␣,mPR␤ and mPR␥—their biochemistry. To date, the an integral membrane (mPR) that binds biochemical characterization of mPR␣,mPR␤ and mPR␥ has progesterone at the cell surface and rapidly generates intracel- involved their expression in either vertebrate cell culture or lular second messengers [4]. This mechanism for progesterone E. coli [19,10,11]. Expression in vertebrate cell culture has been response has been called non-genomic signaling because it fruitful, however, conflicting results were obtained by differ- does not absolutely require transcription to proceed, although ent groups [17,19]. Moreover, this approach is limited by the there is no reason to suspect that non-genomic mechanisms aforementioned overabundance of progesterone-binding pro- do not also regulate transcription [5]. The existence of mPR is teins in vertebrate cells that make data interpretation difficult. a matter of intense debate. Indeed, it has been argued that On the other hand, E. coli do not contain known progesterone there is no need to invoke the existence of a distinct mPR receptors and heterologous expression in this organism has because the nPR itself is capable of mediating rapid non- been successfully used to confirm progesterone binding to genomic effects by binding to and regulating the activity of isolated mPRs embedded in intact E. coli membranes [10,11]. other signaling proteins [6–9]. Nevertheless, several candidate However, while expression in E. coli is certainly useful for mPRs have emerged and, while it is clear that some of these studying progesterone binding to these receptors, it cannot candidates bind progesterone, it is not clear how, or even if, yet be used to investigate functionality, since E. coli it is not they function as receptors to mediate the non-genomic effects known if PAQR receptors function the same in this organism of progesterone. as they do in eukaryotes. Recently, a series of seminal papers were published iden- Thus, a resolution to the debate about whether mPR␣, tifying three integral membrane proteins from fish that not mPR␤ and mPR␥ can sense and respond to progesterone only bind progesterone, but also seem to mediate many of requires a system with two fundamental properties. First, its rapid non-genomic effects [10,11]. These three proteins, such a system must be devoid of known progesterone- renamed mPR␣,mPR␤ and mPR␥, are conserved in vertebrates binding/sensing proteins so that the mPRs can be studied in and belong to a newly characterized family of proteins that isolation. Second, this system must have an intact signaling includes the yeast osmotin receptor (Izh2p) [12] and human apparatus that allows for monitoring signal transduction in adiponectin receptors (AdipoR) [13]. This family is known as response to progesterone. Herein we report the development the progestin- and AdipoQ-Receptor (PAQR) family [14]. of such a system that entails the heterologous expression of A series of follow-up studies produced compelling evidence mPR␣,mPR␤ and mPR␥ in the yeast Saccharomyces cerevisiae. that mPR␣,mPR␤ and mPR␥ function as membrane proges- This choice of model systems is advantageous for several rea- terone receptors. However, as a recent review in this journal sons. First, yeast is a eukaryotic system for which simple yet demonstrates [15], the field remains embroiled in controversy. powerful genetic tools exist. Second, yeast possesses receptors The primary reason for this is the fact that a recent study was in the PAQR family suggesting that the machinery required to unable to reproduce the original results [16,17]. Another signif- read second messengers produced by these proteins is present icant source of contention is the assertion that these receptors [27]. Third, S. cerevisiae neither makes nor uses progesterone. function as a new class of G-protein-coupled receptor (GPCR) In fact, a recent publication showed that massive doses of [18,19,10,11] despite the fact that no other class of PAQR seems progesterone (1 mM) did little more than weakly induce the to couple to G-proteins and that members of the PAQR family general stress–response [28]. The low background biological of receptors bear only superficial similarity to GPCRs. activity of progesterone in this system makes it an ideal model Further investigation is needed before mPR␣,mPR␤ and for the study of individual progesterone receptors in a living mPR␥ can be universally accepted as membrane progesterone cell. Indeed, S. cerevisiae has already been successfully adapted receptors. One side of this equation involves the study of the for the biochemical characterization of nuclear progesterone physiological roles of these proteins in vivo. To date, no stud- receptors [29]. ies have demonstrated an unequivocal role for these proteins We recently published a study showing that the yeast in the physiology of progesterone. Part of the reason for this osmotin receptor (Izh2p) controls a signaling pathway in S. is the overabundance of progesterone-binding proteins and cerevisiae that negatively regulates the expression of a gene putative progesterone receptors in vertebrates that can poten- called FET3 [30]. The physiological importance of this reg- tially confound data analysis. Not only do vertebrate cells ulation is neither clear nor is it actually important in the possess classical nuclear progesterone receptors, they also context of this investigation. What is important is that we possess the other members of the nuclear hormone recep- also showed that activation of human adiponectin receptors, tor family for which progesterone may function as either an when heterologously expressed in yeast, had the same effect agonist or antagonist [20,21]. Vertebrate cells also contain a on FET3 expression. The regulation of the same pathway by variety of other progesterone-binding proteins that have been fungal and human PAQRs suggests that PAQRs from diverse proposed to function as mPRs [22,23]. Progesterone has even sources generate similar second messengers when expressed been shown to function as an allosteric regulator of a vari- in yeast. The identity of this second messenger and the mech- ety of enzymes and receptors [24–26]. Thus, it is difficult to anism of signal transduction for PAQRs in yeast are still under 1162 steroids 73 (2008) 1160–1173

investigation and these topics are beyond the scope of this ing frames by gap repair. Primers for all cloning reactions are study. listed in Supplemental Data. Proteins expressed in pYES260 Herein, we demonstrate that the expression of the FET3 have an N-terminal 6×-histidine tag followed by a TEV pro- gene can be used as a general reporter of the functionality of tease cleavage site. Proteins expressed in pGREG536 have human PAQR receptors. Using this reporter system to study an N-terminal 7×-HA tag. Proteins expressed in the pRS316 the mPRs, we made several important findings. First and fore- vector are untagged. The pYES260, pRS316 and pGREG536 vec- most, we demonstrated mPR␣,mPR␤ and mPR␥ repress FET3 tors allow for galactose-inducible expression via the GAL1 in response to progesterone while other PAQR receptors do promoter. We see no vector-dependent difference in the func- not. This confirms beyond reasonable doubt that these pro- tionality of any PAQR receptor, indicating that receptors in teins can sense and respond to progesterone. Moreover, the this family seem to be highly tolerant of N-terminal modifi-

ED50 values for the activation of the mPRs by progesterone cations. The FET3-lacZ plasmid was previously described [30]. demonstrate that the mPRs are most responsive to proges- Plasmids derived from pADM4 carrying hyperactive alleles of terone at physiologically relevant concentrations. Second, we Gpa1p (Gpa1pQ323L) [38] and Gpa2p (Gpa2pQ300L) [39] driven show that this assay can be used to probe the agonist profiles by the ADH1 promoter were kindly provided by Dr. Henrik for these receptors. Not only do these receptors have specifici- Dohlman (UNC Chapel Hill). A pRS316-based plasmid contain- ties that are distinct from nPR, they are activated by ligands, ing the STE2 gene under the control of the GAL1 promoter suchas17␣-hydroxyprogesterone, that are known to activate [40] was kindly provided by Dr. Nicholas G. Davis (Wayne State non-genomic pathways but not nPR [3]. We also demonstrate University). that , an important antagonist of nuclear pro- gesterone receptors, actually serves as a weak agonist of the 2.3. Assays and growth conditions mPRs, a fact that is consistent with what is known about non-genomic progesterone signaling [31]. Third, we show that Strains were maintained using standard protocol and grown human PAQR6 and PAQR9, both uncharacterized members of in synthetic defined (SD) media with the appropriate amino the PAQR family likely function as additional vertebrate mPRs. acids to match the auxotrophies of the individual strains. Low Accordingly, we renamed them mPR␦ and mPR␧, respectively. iron medium (LIM) contains EDTA to limit iron-bioavailability Finally, we demonstrate that the ability of these proteins to and its composition has been previously described [30]. When 3+ sense and respond to progesterone requires neither human supplemented with only 1 ␮MFe , LIM is considered iron- nor yeast G␣-proteins making it unlikely that the mPRs func- deficient and the FET3 gene is fully induced under these tion as GPCRs in the classical sense. conditions. ␤-Galactosidase (lacZ) assays were performed as previously described [30]. In brief, overnights of cells grown in SD-glucose media were re-inoculated into iron-deficient LIM 2. Materials and methods to induce the expression of FET3. 2% galactose was used as a carbon source to induce full expression of PAQR genes driven 2.1. Yeast strains by the GAL1 promoter, while 0.05% galactose/1.95% raffinose was used for reduced PAQR expression. All ligands were added Wild type BY4742 (Mat ␣), BY4741 (Mat a) and gpa2 to the growth medium upon re-inoculation into LIM. For exper- (Mat ␣, BY4742 background) mutant yeast strains were iments in which steroids were added as ligands, steroids were obtained from Euroscarf (http://web.uni-frankfurt.de/fb15/ added from ethanol stocks and “untreated” controls are actu- mikro/euroscarf/). gpa1 mutants are inviable due to con- ally treated with an equal volume of ethanol to control for stitutive growth arrest caused by the hyperactivation of the vehicle effects. Cells were allowed to grow to mid-log phase at Ste4p/Ste118p G␤␥ subunit in the absence of the Gpa1p G␣ which time the activity of the FET3 gene was monitored using subunit. Viability of gpa1 mutant strains can be restored by a FET3-lacZ promoter-reporter construct. lacZ activities are concomitant deletion of the Ste7p MAP kinase that functions presented as a percentage of activity seen in untreated cells downstream of Ste4p/Ste18p. The gpa1ste7 mutant strain expressing the appropriate empty expression vector control. (in the BY4741 background) was kindly provided by Dr. Henrik For individual experiments, each data point has been done in Dohlman (University of North Carolina, Chapel Hill) [32]. The triplicate and the error bars represent ±1 S.D. All experiments STY50 strain lacking the HIS4 gene [33] was kindly provided by were performed at least three times and a representative

Dr. Gunnar von Heijne (Stockholm University). experiment is shown. EC50 values were obtained using the web-based BioDataFit software using sigmoidal curve-fitting 2.2. Plasmids and primers (http://www.changbioscience.com/stat/ec50.html). Total preparations were prepared as Human PAQRs were cloned into various vectors by PCR from previously described [30]. Protein concentrations were deter- commercially available cDNAs, the source and names of which mined and equal amounts were loaded onto SDS-PAGE gels are listed in Supplemental Data. PAQRs were cloned into the for Western blotting. Western blots were performed using pYES260 [34], pRS316 [35] or pGREG536 [36] expression vectors standard protocol with a rabbit polyclonal anti-HA primary using gap repair. The pJK90 plasmid containing a dual topol- antibody (Santa Cruz Biotechnology) and a goat anti-rabbit ogy reporter (DTR)-tagged OST4 gene driven by the constitutive IgG–HRP conjugate as the secondary antibody (Santa Cruz TPI1 promoter was kindly provided by Dr. Gunnar von Hei- Biotechnology). Proteins were detected by chemilumines- jne (Stockholm University) [37]. The OST4 open reading frame cence. Determination of glycosylation with EndoH treatment in pJK90 was replaced with the PAQR6 and mPR␣ open read- was performed as published [41]. steroids 73 (2008) 1160–1173 1163

2.4. Sequence analysis

Multiple sequence alignments and bootstrapped phylogenetic trees were produced using ClustalX with default parameters [42]. Trees were visualized using Tree View [43] and TreeView X(http://darwin.zoology.gla.ac.uk/∼rpage/treeviewx/index. html). Pairwise sequence analysis was performed at the NCBI website by Blast 2 Sequences [44]. Hydropathy plots were first generated with TopPredictII 1.2 [45] using the Kyte–Doolittle algorithm and default parameters. Data was downloaded into a spreadsheet and the hydropathy values were aligned based on the multiple sequence alignment produced by ClustalX. Average hydropathy across the entire set of aligned proteins was then calculated. Sequences used for these comparisons are listed in Supplemental data. Most sequences were obtained from the NCBI database, however, sequences from Trichoplax adhaerens, Nematostella vectensis, Lottia gigantea, Capitella sp., Branchiostoma floridae and Ciona intestinalis were obtained from the Joint Genome Institute website (http://genome.jgi-psf.org/euk home.html).

3. Results

3.1. Heterologous expression of human mPRs in yeast

When grown in iron-deficient medium, the FET3 gene is induced to facilitate the uptake of exogenous iron. We previously demonstrated that the yeast osmotin receptor (Izh2p) activated a pathway that resulted in the constitutive repression of FET3 in iron-deficient conditions [30]. Control experiments demonstrated that this effect was not an artifact of the FET3-lacZ reporter and that the expression of FET3 was, indeed, regulated by Izh2p. We also demonstrated that the human adiponectin receptors (AdipoR1 and AdipoR2) could be functionally expressed in yeast. In these experiments, expres- sion of the FET3 gene responded reciprocally to the amount of adiponectin in the medium in cells expressing AdipoR1 Fig. 1 – Functional expression of human mPR␣,mPR␤ and and AdipoR2 [30]. The EC50 values for adiponectin were 0.7 ␥ and 2.4 pM for AdipoR1 and AdipoR2, respectively (Table 1). mPR in yeast. All cells are wild type and are grown in This data is re-plotted in Fig. 1A to demonstrate two impor- iron-deficient LIM. Medium in panels (A) and (B) contains tant concepts. First, the expression of the FET3 gene can be 0.05% galactose/1.95% raffinose and medium in panel (C) used as a reporter for the activity of human PAQR recep- contains 2% galactose. In all boxes, the activity of the FET3 ␤ tors. Second, PAQR receptors from diverse sources activate the gene is monitored by measuring -galactosidase activity same pathway in yeast. This led us to predict that this sys- produced by the FET3-lacZ reporter. (A) All PAQRs are cloned into the pYES260 vector except AdipoR2, which is cloned into pGREG536. White symbols show the effect of progesterone on FET3 in cells carrying either empty ␣ ␤ Table1–EC50 values for various PAQR ligands expression vector or vectors that express mPR ,mPR or mPR␥. Grey symbols show the effect of adiponectin on FET3 log EC50 pM (EC50) in cells carrying either empty expression vector or vectors Adiponectin Progesterone that express AdipoR1 or AdipoR2. (B) The effect of various ␥ AdipoR1 −0.13 ± 0.08 (0.7 pM) N.R. steroids on the FET3 gene in cells expressing either mPR AdipoR2 0.38 ± 0.17 (2.4 pM) N.R. or mPR␣ from the pYES260 plasmid. (C) The effect of mPR␥ N.R. 3.11 ± 0.44 (1.3 nM) various steroids on FET3 in cells expressing mPR␣ from the PAQR6 N.R. 3.42 ± 0.88 (2.6 nM) pRS316 plasmid. When data points overlap or are very ␣ ± mPR N.R. 3.36 0.08 (2.3 nM) close to overlapping, combined symbols are used. mPR␤ N.R. 3.21 ± 0.61 (1.6 nM) PAQR9 N.R. 4.14 ± 0.86 (13.8 nM)

N.R., no response. 1164 steroids 73 (2008) 1160–1173

Table2–EC50 values for various PAQR ligands

log EC50 nM (EC50)

mPR␥ mPR␣

Progesterone 0.04 ± 0.25 (1.1 nM) 0.34 ± 0.08 (2.2 nM) 17␣-Hydroxyprogesterone 0.97 ± 0.63 (9.3 nM) 1.01 ± 0.44 (10.2 nM) RU-486 3.83 ± 0.23 (6.8 ␮M) 4.04 ± 0.26 (11.0 ␮M) Progesterone + 10 ␮M testosterone 1.03 ± 0.53 (10.7 nM) N.D. Progesterone + 100 nM RU-486 1.19 ± 0.12 (15.5 nM) N.D.

N.D., not determined. tem could be used to address whether mPR␣,mPR␤ and mPR␥ Mifepristone, also known as RU-486, is a clinically impor- could sense and respond to progesterone. We cloned all three tant antagonist of the nuclear progesterone receptor [47].We human receptors into GAL1-driven expression plasmids and examined the ability of this synthetic steroid to antagonize grew them in iron-deficient LIM containing 0.05% galactose. mPR␥ and mPR␣. Surprisingly, RU-486 functioned as a weak

In cells expressing the mPRs, the expression of the FET3 gene agonist of both receptors with EC50 values in the low ␮M range responded reciprocally to progesterone in a dose-dependent (Fig. 2B and Table 2). RU-486 had no effect on yeast carrying manner (Fig. 1A). The EC50 values for progesterone were 1.3, 2.3 empty expression vector (Fig. 2B). We also demonstrated that and 1.6 nM for mPR␥,mPR␣ and mPR␤, respectively (Table 1). RU-486 functions as a weak antagonist of mPR␥ at lower con- Wild type cells carrying empty expression plasmid did centrations. Fig. 2C shows that RU-486 weakly antagonizes not respond to either adiponectin or progesterone, indicat- mPR␥ in the presence of 100 nM progesterone and that this ing that the effects described above cannot be attributed to effect disappears at higher concentrations of RU-486. Further- an endogenous yeast protein, such as a yeast PAQR recep- more, exposure of cells to 100 nM RU-486 causes a 15-fold tor. Furthermore, AdipoR1 and AdipoR2 do not respond to increase in the EC50 of progesterone for the activation of mPR␥ progesterone at concentrations as high as 10 ␮M(Fig. 4A), (Fig. 2A and Table 2). demonstrating that progesterone specifically affects mPR␣, mPR␤ and mPR␥ and does not generally affect yeast expressing 3.3. Sequence analysis of the mPR family non-native PAQR receptors. The contains 11 genes encoding proteins in 3.2. Specificity of mPR˛, mPRˇ and mPR the PAQR family [14], all of which are highly conserved in the vertebrate lineage. PAQR5, PAQR7 and PAQR8 encode the We also investigated the steroid specificity of mPR␣ and mPR␥ putative membrane progesterone receptors and their gene and found that 17␣-hydroxyprogesterone was an effective products have been named mPR␥,mPR␣ and mPR␤, respec- activator of both receptors, albeit with a lower EC50 (Fig. 1B, tively. A bootstrapped phylogenetic tree (Supplemental Fig. Table 2). A further analysis of mPR␣ revealed that the recep- 1A) shows the relatedness of the mPRs to other receptors in tor was largely permissive of alterations at the 17 and 21 the PAQR family including the human adiponectin receptors positions of the pregnane ring. 17␣-Hydroxyprogesterone, and the yeast osmotin receptor homologues, Izh1p, Izh2p and 21-hydroxyprogesterone and 17␣,21-dihydroxyprogesterone Izh3p. We included sequences from a variety of species to were all similarly effective activators of mPR␣. However, demonstrate conservation throughout the vertebrate lineage. 11␤,17␣,21-trihydroxyprogesterone (cortisol) was an ineffec- This tree clearly demonstrates that the PAQR family can be tive agonist (Fig. 1C). grouped into three distinct clades. Human PAQR10 and PAQR11 While testosterone is closely related in structure to pro- belong to a highly divergent clade of PAQRs that contains bac- gesterone, it is a poor agonist of the non-genomic pathways terial proteins previously characterized as hemolysins [48]. of progesterone signaling [46]. However, testosterone does We designated this clade as Class III and used it as an out- seem to competitively inhibit progesterone binding to mPR␥ group to root the tree. A second clade includes human PAQR1, and mPR␣ in mammalian cells [19,10,11] and our data sug- PAQR2, PAQR3 and PAQR4 as well as the yeast PAQR homo- gests that mPR␥ and mPR␣ are tolerant to substitutions at logues. We designated this clade as Class I. Homologues of the 17 position of the pregnane ring where testosterone and PAQR1, PAQR2 and PAQR3 can be found in fungi. A homologue progesterone differ. This raised the possibility that testos- of PAQR4 can be found in N. vectensis, but not T. adhaerens, sug- terone might function as an antagonist of the mPRs. Fig. 1B gesting that this gene may have originated in eumetazoans. shows that testosterone had no agonist activity against either The three mPRs belong to a third clade designated as Class II. mPR␥ or mPR␣ in concentrations up to 10 ␮M. However, This tree indicates that Class II receptors can be subdivided when the response of mPR␥ to progesterone was measured into two subgroups—one that contains mPR␥ and one that in the presence of 10 ␮M testosterone, the EC50 of proges- contains mPR␣ and mPR␤. In addition to mPR␥, the human terone increased 10-fold (Fig. 2A and Table 2). Exposure genome encodes two additional PAQRs (PAQR6 and PAQR9) of cells to 10 ␮M estradiol had no effect on the EC50 of that belong to the mPR␥ subgroup. We could not identify a progesterone, indicating that this effect was specific for testos- PAQR6 homologue in chicken or an mPR␣ homologue in frog. terone and not a non-specific effect of steroids on the assay The absence of such sequences in the databases may be due system (Fig. 2A). to gaps in G. gallus, X. tropicalis or X. laevis genomes, however, steroids 73 (2008) 1160–1173 1165

brates contain only one copy of each of the mPRs, teleost fish genomes (herein represented by zebrafish) encode three distinct mPR␥ isoforms, two of which are 100% identical and found in tandem on 7. The zebrafish genome also encodes multiple paralogs of mPR␣, AdipoR1, PAQR4 and PAQR10. The multiple PAQR paralogs in zebrafish fish are con- sistent with the genome duplication event that is believed to have led to the evolution of teleost fish [49]. The mPRs have been proposed to be a novel class of GPCR. Another bootstrapped phylogenetic tree (Supplemental Fig. 1B) that contains the human and yeast PAQRs as well as several proteins belonging to the GPCR [50] and alkaline ceramidase [51] protein families. The tree is rooted with the GPCRs as an outgroup. The PAQRs, GPCRs and alkaline ceramidases are alike in that all three families have a core of seven trans- membrane (TM) domains. This tree demonstrates that the PAQR family is no more similar to GPCRs than they are to alkaline ceramidases, suggesting that any structural similarity between the PAQR and GPCR families is superficial. It should be noted, however, that the GPCR family is highly divergent and that there are no amino acid motifs that unify the entire family [50]. Thus, it is still possible that the PAQRs represent a unique class of highly divergent GPCR. Fig. 3 shows a multiple sequence alignment of the five human Class II PAQRs. The entire PAQR family is unified by the presence of seven predicted TMs and three con- served regions (see alignment in Ref. [27]). First, there is a conserved motif that precedes TM1. This motif has the con-

sensus PxnGYRxnEx2Nx3H, although this motif is truncated to Ex2–3Nx3H in Class III PAQRs. A second motif spans the end of TM2 and the beginning of TM3 and has the consen-

sus sequence Sx3HxnD. A third motif spans the loop preceding TM7 and has the motif PEx3PGxnHQx2H, although this is also truncated to Hx3H in Class III proteins. The seven TM core and these three short motifs are all that unify the entire PAQR fam- ily. Class II PAQR receptors are unique in that they contain an eighth predicted hydrophobic motif that is C-terminal to the conserved PAQR core (see Fig. 5 for hydropathy plots). Fig. 2 – Steroid specificities for mPR␥ and mPR␣.Inall cases, FET3 expression is measured using the FET3-lacZ 3.4. Effect of progesterone on Class II receptors reporter. All PAQRs are cloned into the pGREG536 vector. All cells are wild type and are grown in iron-deficient LIM mPR␥ is more similar to PAQR6 and PAQR9 than it is to mPR␣ containing 0.05% galactose/1.95% raffinose. (A) The and mPR␤. Since mPR␥,mPR␣ and mPR␤ all function as pro- dose–response of FET3 in cells expressing mPR␥ plasmid to gesterone receptors, it is likely that PAQR6 and PAQR9 do as progesterone either alone or in the presence of 10 ␮M well. To test this, we cloned all 11 human PAQRs into GAL1- ␤-estradiol (E), 10 ␮M testosterone (test) or 100 nM RU-486 driven yeast expression vectors to determine if they could (RU). (B) Dose–response of FET3 to either progesterone (P) or repress FET3 in response to progesterone treatment (We have RU-486 (RU) in cells carrying either empty expression vector evidence for expression of all human PAQRs except PAQR10, or vectors that express mPR␥ or mPR␣. (C) Dose–response data not shown). Fig. 4A shows that 10 ␮M progesterone was of FET3 to RU-486 in cells carrying either empty expression an effective agonist of all five Class II PAQRs, but was ineffec- vector or a vector that expresses mPR␥. Cells are either tive against receptors in Classes I and III. The dose–response treated with RU-486 alone or in the presence of 100 nM of PAQR6 and PAQR9 to progesterone reveals EC values of 2.6 progesterone (P). When data points overlap or are very 50 and 13.8 nM, respectively (Fig. 4B and Table 1). Fig. 4B shows close to overlapping, combined symbols are used. that, like mPR␥ and mPR␣, PAQR6 and PAQR9 can be activated by 17␣-hydroxyprogesterone, but not testosterone. we could find neither supporting cDNAs nor ESTs, which sug- gests that these genes may have been lost in these species. 3.5. Structural analysis of Class II PAQRs The PAQR9 gene in the G. gallus genome is interrupted by an unsequenced gap. Therefore the full sequence is not shown In Fig. 5A, overlaid hydropathy plots of various vertebrate in Supplemental Data. While the genomes of higher verte- homologues of the human adiponectin receptors demonstrate 1166 steroids 73 (2008) 1160–1173

Fig. 3 – Multiple sequence alignment of human Class II PAQRs. This alignment was originally performed using ClustalX but was modified manually afterwards. TM and loop regions (L) are numbered. Predicted TMs are also boxed. Black shading shows amino acids that are highly, but not universally, conserved in the entire PAQR family. Grey shading indicates amino acids that are conserved in all vertebrate members of the mPR␥/PAQR6 clade, although only human sequences are shown. Circled amino acids indicate the positions of intron/exon boundaries in the pre-mRNA. Arrow indicates the location of truncations in the mPR␥ and mPR␣ proteins discussed in Fig. 6.

the seven predicted TMs of the conserved PAQR core. The the same method that was previously used to determine the same workup for vertebrate proteins in the mPR␣/␤ clade topology of the yeast Izh2p receptor [41]. In this method, an (Fig. 5B) and the mPR␥ clade (Fig. 5C and D) clearly shows HA-Suc2p-His4p tag is placed at the C-terminus of a protein of that the seven hydrophobic domains of the PAQR core are interest (Fig. 5F). The HA (hemagglutinin) part of the tag allows conserved with varying degrees of hydrophobicity. Topolog- for detection of the chimera by Western blot. The His4p part ical studies of the yeast and human Class I receptors as of the tag encodes a functional histidinol dehydrogenase that well as bacterial Class III receptors indicated that the C- is required for histidine biosynthesis. This enzyme requires terminus of these receptors is extracellular [52,53,41]. This cytosolic NADH as a cofactor and will only rescue the histi- led to the topological model shown in Fig. 5E showing the dine auxotrophy of a his4 mutant strain if the tag is cytosolic. conserved PAQR core and the location of the three highly con- In addition, if the C-terminus is extracellular, it must pass served motifs shown in Fig. 3. If the additional hydrophobic through the lumen of the endoplasmic reticulum. The Suc2p domain at the C-termini of the Class II receptors is, indeed, fragment of the tag contains multiple N-glycosylation sites, a TM, then the C-termini of receptors in this class should be which will be glycosylated if it passes through the ER lumen. intracellular. This modification can be detected by a shift in molecular To test this, we used the dual topology reporter method weight in Western blots after treatment with an endoglycosi- to probe the topology of mPR␣ and PAQR6 in yeast. This is dase. steroids 73 (2008) 1160–1173 1167

on the ability of these receptors to sense and respond to pro- gesterone (Fig. 6B).

3.6. Lack of involvement of G˛-proteins in mPR-dependent signaling

It has been proposed that mPR␣,mPR␤ and mPR␥ function as a novel class of GPCR [18,19,10,11]. If this were true, then heterotrimeric G-proteins would be required as intracellular second messengers downstream of the mPRs. Yeast possess only two heterotrimeric G␣␤␥-protein complexes. The first includes Gpa1p (␣), Ste4p (␤) and Ste18p (␥) and is coupled to the Ste2p mating pheromone-sensing GPCR [32]. The sec- ond is less well-characterized and contains Gpa2p (␣) and a non-canonical ␤-subunit, Acs1p. To date, no ␥-subunit has been identified for Gpa2p. Gpa2p is coupled to the Gpr1p glucose-sensing GPCR [54]. Both mPR␥ and mPR␣ were capa- ble of sensing and responding to progesterone in strains Fig. 4 – Identification of additional membrane progesterone lacking either Gpa1p or Gpa2p (Fig. 6C). In addition, we demon- receptors. In all panels, cells were grown in medium strated that neither overexpression of the Ste2p GPCR from ␣ containing 0.05% galactose/1.95% raffinose and FET3 the GAL1 promoter nor its concomitant activation with - activity is measured using the FET3-lacZ construct as a factor pheromone could recapitulate the effect of the PAQRs on reporter. All PAQRs are cloned into the pYES260 vector the FET3 gene (Fig. 6D). Moreover, expression of constitutively Q323L Q300L except AdipoR2, which is cloned into pGREG536. (A) active alleles of Gpa1p (Gpa1p ) and Gpa2p (Gpa2p ) Response of FET3 in cells expressing all 11 human PAQR were incapable of causing repression of FET3 (Fig. 6E). proteins to 10 ␮M progesterone. (B) Response of FET3 to various steroids in wild type cells expressing either PAQR6 4. Discussion (white symbols) or PAQR9 (grey symbols).

The definitive classification of mPR␣,mPR␤ and mPR␥ as membrane progesterone receptors is still a matter of debate. Consistent with the model shown in Fig. 5E, C-terminally The recent publication of conflicting data that directly chal- tagged Izh2p was previously shown to be unable to con- lenged the role of mPR␣,mPR␤ and mPR␥ in progesterone fer histidine prototrophy, indicating that its C-terminus was signaling [17] has reinforced the controversy. To address extracellular (see Ref. [41]). When this tag was placed at the whether or not the mPRs are, indeed, membrane progesterone C-terminus of PAQR6 and mPR␣, the resulting chimeras could receptors, we expressed the mPRs in the tractable eukary- still respond to progesterone demonstrating that the tag did otic model organism, S. cerevisiae. The obvious reason for this not affect functionality (Fig. 5G). Moreover, both chimeras choice is that yeast do not make or use progesterone. Indeed, could weakly rescue the histidine auxotrophy of a his4 strain, that progesterone has very low biological activity towards indicating that their C-termini are intracellular (Fig. 5H). Treat- this organism [28] and that its genome does not encode pro- ment of membrane extracts from cells expressing PAQR6 and teins in the nuclear receptor family [55] make S. cerevisiae an mPR␣ with the endoglycosidase, EndoH, does not alter the ideal model system to study progesterone receptors. In fact, mobility of either PAQR6 or mPR␣ in Western blots (Fig. 5I). recombinant yeast is routinely used to study the structure and A change in mobility for RNaseB–aprotein that is known to function of vertebrate nuclear progesterone receptors [29]. be N-glycosylated – is shown as a positive control. These find- Data from previous publicaitons demonstrate that Izh2p, ings indicate that the C-terminus of PAQR6 and mPR␣ does not an endogenous yeast PAQR receptor, activates a pathway that pass through the ER lumen and that the C-terminus of Class leads to the repression of a gene called FET3 [30]. The phys- II PAQRs does, indeed, contain an additional TM. iological significance of this regulation, while an important Thus, human Class II receptors are unified by the presence aspect of fungal biology, is irrelevant for this discussion, which of an additional TM that is C-terminal to the conserved PAQR focuses solely on the development of this phenomenon into core and by the fact that all five respond to progesterone when an assay for human PAQR receptor activation. To demonstrate expressed in yeast. This led us to postulate that this addi- proof of principle, we heterologously expressed the human tional hydrophobic domain is responsible for progesterone adiponectin receptors, AdipoR1 and AdipoR2, in yeast and sensing. Unfortunately, there are no amino acids in this TM showed that they repress FET3 in an adiponectin-dependent that are conserved in all five receptors (see Fig. 3). Hence, we manner [30]. The fact both yeast and human PAQRs repress did not undertake site directed mutagenesis to determine if FET3 in yeast suggests that PAQR receptors from various any particular amino acid is involved in sensing. Instead, we sources generate the same second messenger, which regulates truncated the entire TM for mPR␥ and mPR␣. Western blots the pathway leading to FET3 repression. It is important to state show that these truncation mutants are stably expressed in that we are only suggesting that receptors in the PAQR fam- yeast (Fig. 6A). Unexpectedly, these truncations had no effect ily generate similar second messengers, not that the signaling 1168 steroids 73 (2008) 1160–1173

Fig. 5 – Topological analysis of Classes I and II PAQRs. (A–D) Hydropathy plots for the individual proteins analyzed in Fig. 3 were generated and aligned (dotted lines). An average hydropathy plot for all members of each clade was generated (solid lines). The core PAQR motif is shaded in grey and the predicted TMs are numbered. All vertebrate members of the AdipoR1 and AdipoR2 clade (A), the mPR␥/PAQR6 clade (B), the mPR␣/mPR␤ clade (C) and the PAQR9 homologues (D). (E) Predicted topology of the core PAQR motif with the locations of the three highly conserved motifs shaded in black in Fig. 4.The predicted eighth TM in the mPRs is shown with a dotted line. (F) The structure of the dual topology reporter tag. (G) DTR-tagged PAQR6 and mPR␣ expressed in the pJK90 plasmid repress FET3-lacZ in response to 100 nM progesterone in cells grown in medium containing 0.05% galactose/1.95% raffinose. (H) Rescue of the histidine auxotrophy of the STY50 strain by DTR-tagged PAQR6 and mPR␣ on plates containing histidinol. Plasmids expressing untagged versions of PAQR6 and mPR␣ are shown as negative controls. A plasmid expressing DTR-tagged Ost4p is shown as a positive control. (I) Membrane extracts from cells expressing DTR-tagged PAQR6 or mPR␣ are either left untreated (−) or treated with the endoglycosidase EndoH (+) and subsequently run on protein gels and transferred to nitrocellulose membranes for Western blot. Anti-HA antibodies were used to detect the DTR tag. (Left) Coomassie stained gel of EndoH treated RNase B under the same conditions as those used to treat the cell membrane extracts is shown on the right as a positive control. steroids 73 (2008) 1160–1173 1169

Fig. 6 – Truncation mutations and G-protein signaling. In all cases, FET3 expression is measured using the FET3-lacZ reporter. (A) Full length and truncated mPR␥ and mPR␣ were expressed in wild type cells grown in 2% galactose from the pGREG536 vector. The location of the C-terminal truncations is shown in Fig. 3. All proteins possess an N-terminal 7×-HA tag. Proteins were detected by Western blot using an anti-HA antibody. (B) The dose–response of FET3 to progesterone in cells expressing the full length or truncated mPR␥ and mPR␣ and grown in 0.05% galactose/1.95% raffinose. (C) The ability of mPR␣ and mPR␥ to respond to progesterone and repress FET3 is not impaired in either gpa2 or gpa1 cells (gpa1 cells also lack the STE7 gene, see text). (D) Overexpression of the Ste2p GPCR using the GAL1 promoter does not repress FET3 in wild type cells (mat a) grown in 2% galactose. Activation of overexpressed Ste2p via the addition of 1 ␮M of its agonist, ␣-factor, also has no effect on FET3 under these conditions. (E) Expression of constitutively active alleles of Gpa1p (Gpa1pQ323L) and Gpa2p (Gpa2pQ300L) from the GAL1 promoter had no effect on FET3 in cells grown in 2% galactose.

components and pathways downstream of PAQR receptors are by any PAQR receptor. These results unambiguously demon- conserved from yeast to humans. We are in the process of strate that mPR␣,mPR␤ and mPR␥ are capable of conferring mapping the signaling components downstream of Izh2p in progesterone-responsiveness onto yeast cells. When coupled yeast and future research may yet reveal such conservation. In with previous studies showing progesterone binding by mem- the meantime, we used the FET3 gene as a reporter to address branes from E. coli expressing mPR␣,mPR␤ and mPR␥, our two highly contentious issues surrounding mPR␣,mPR␤ and data strongly argue that these proteins directly mediate the mPR␥. First and foremost, we addressed whether or not mPR␣, response to progesterone. That said, we cannot yet rule out mPR␤ and mPR␥ actually function as progesterone receptors. the remote possibility that yeast express an unidentified Second, we began to address models for how the mPRs convert progesterone-binding protein that interacts with the mPRs extracellular progesterone into intracellular second messen- and functions as the ligand-binding component. gers. The EC50 for progesterone activation of mPR␣,mPR␤ and Our data clearly demonstrate that mPR␣,mPR␤ and mPR␥ in yeast is between 1 and 3 nM, values that are consistent ␥ ∼ mPR , when heterologously expressed in yeast, mediate with the Kd’s for progesterone binding to mPRs ( 5 nM) deter- progesterone-dependent repression of the FET3 gene. Con- mined by Thomas [56] These EC50 values are also close to the trol experiments showed that progesterone did not affect physiological concentration of progesterone in human serum, FET3 in yeast carrying empty expression vector or the human which has been estimated to be between 1 and 10 nM in men adiponectin receptors, indicating that this effect was not (a) a and non-pregnant women in the follicular phase of the men- non-specific effect of progesterone, (b) the result of expressing strual cycle [57,58]. Thus, our data suggests that human mPR␣, a foreign membrane protein or (c) a general effect mediated mPR␤ and mPR␥ are most responsive to progesterone at phys- 1170 steroids 73 (2008) 1160–1173

iologically relevant hormone concentrations and would likely now this model seems to be either universally accepted or at function as legitimate progesterone receptors. This interpre- least not seriously questioned. A major problem with the GPCR tation must be tempered by the possibility that other human hypothesis is that there is no evidence for the involvement proteins, not present in the yeast system, may modulate the of G-proteins in signaling by any other class of PAQR recep- response of these receptors to progesterone. tor, suggesting that either there are significant differences Analysis of the steroid specificity of mPR␣ and mPR␥ between PAQR classes or the GPCR model needs revision. activation indicates that they have distinct agonist profiles Before discussing our data, it is necessary to discuss the evi- from nuclear progesterone receptor and provides evidence dence for G-protein involvement. First, there is a large body of that their profiles are similar to those of the receptors that evidence suggesting that Gi␣ proteins are involved in some of mediate non-genomic responses of progesterone [46,3,31].To the non-genomic effects of progesterone [10,11]. The involve- begin with, our data indicate that the steroid activation pro- ment of Gi␣ in progesterone signaling naturally led to the file of the mPRs correlates well with a study that showed hypothesis that the hypothetical mPR was a GPCR. The dis- 17␣-hydroxyprogesterone (17␣-HP), 21-hydroxyprogesterone covery that mPR␣,mPR␤ and mPR␥ belonged to a family of (21-HP) and 17␣,21-dihydroxyprogesterone (17␣,21-DHP) were proteins with seven TMs and that these receptors had intracel- effective agonists of the non-genomic progesterone signaling lular C-termini bolstered this idea. More compelling evidence pathway but 11␤,17␣,21-trihydroxyprogesterone (cortisol) and for the classification of mPRs as GPCRs came from experi- testosterone were not [46,3]. Another distinction between the ments showing co-immunoprecipitation of a Gi␣-protein with mPRs and nPR is the fact that mifepristone (RU-486), a potent mPR␣ [18,19]. In addition, Thomas et al. showed that dele- nPR antagonist, functions as a weak agonist for both mPR␥ and tion of the soluble part of the C-terminus of mPR␣ abrogated ␣ ␮ 35 ␥ mPR at high concentrations RU-486 (EC50 >5 M) and, enig- the stimulation of S- -GTP to Gi␣ in response to progestin ␥ matically, as only a very weak antagonist of mPR at lower treatment [19], suggesting that activation of Gi␣ requires the concentrations (100 nM), shifting the EC50 for progesterone C-terminus of mPR␣ in a manner that is reminiscent of GPCRs. from ∼1to∼15 nM. Closer scrutiny of these findings reveals that none The ability of 17␣-HP and mifepristone to activate mPRs offer unequivocal evidence that the mPRs are GPCRs. is particularly intriguing because the former steroid has very First, co-immunoprecipitation experiments with membrane- low agonist activity towards nPR [46] and the latter is actually associated proteins can be misleading since one is not actually an antagonist of nPR [47]. Both, however, seem to be able to precipitating a protein, but the entire membrane complex in ␣ activate non-genomic signaling [3,31]. In fact, the differential which the protein is embedded. Since both mPR and Gi␣ reactivity of these steroids towards the genomic and non- associate with membranes [61], it is not surprising that they genomic pathways has been used as evidence for the existence co-precipitate. Consequently, a direct interaction between Gi␣ of distinct membrane progesterone receptors [3]. Hence, the and mPR is yet to be unambiguously demonstrated and the specificity profiles of the mPRs in yeast indicate that these possibility that the involvement of Gi␣ in progesterone signal- receptors closely resemble the hypothetical non-genomic pro- ing is indirect has not yet been ruled out. Second, we feel that gesterone receptors. the issue of receptor topology has introduced bias towards the The unique agonist profiles for the mPRs are also intriguing characterization of these receptors as being GPCRs in such a from a physiological standpoint. While the physiological lev- way that the GPCR model has been accepted without con- els of 17␣-HP, 21-HP and 17␣,21-DHP are low in human serum crete proof. Simply having seven TMs and an intracellular (∼1 nM) they can be as high as 10–20 nM in pregnant women C-terminus does not make a protein a GPCR.

[59]. Considering that the EC50 values for the activation of We have presented data that contradicts the characteriza- mPR␣ by 17␣-HP, 21-HP and 17␣,21-DHP are similar (∼10 nM), it tion of these receptors as GPCRs. To begin with, data in this is possible that these steroids may indeed be physiologically study and from other publications [52,53,41,17,19] support a relevant ligands for this receptor. On the other hand, while model in which PAQRs from all Classes share an identical core testosterone seems to function as an antagonist of mPR␥, the structure of seven TMs with identical topologies and that Class levels of testosterone required for this effect (10 ␮M) seem to II PAQRs possess an eighth TM that places the C-terminus in be far too high to be physiologically important [60]. The phar- the cytoplasm (see Fig. 5E). Our data indicating that all PAQRs macokinetics of RU-486 [47] indicate that low doses of RU-486 activate the same signaling pathway in yeast is consistent result in sustained circulating levels in the 100 nM range where with a model in which all PAQRs have a similar core structure it antagonizes mPR␥, while high doses result in sustained lev- and mechanism of action. To be clear, however, no group has els in the 5 ␮M range, where it agonizes mPR␥. Thus, RU-486 undertaken a rigorous mapping of the number and topology of may have opposing effects on the mPRs depending on the dose TMs in any PAQR. A satisfactory resolution to the debate about given. whether or not the mPRs resemble GPCRs or other PAQRs will The second contentious issue that we attempted to address have to wait for such a study. is the nature of the second messenger produced by the mPRs. Regardless of whether or not the mPRs structurally When first discovered, bioinformatic analysis suggested that resemble GPCRs, our data demonstrate that no human het- the mPRs had seven TMs [10,11]. This naturally led people to erotrimeric G-proteins are essential for mPR-dependent signal postulate that the mPRs were similar to GPCRs despite the transduction in response to progesterone. Since many human fact that the mPRs bear no more similarity to GPCRs than GPCRs have been shown to functionally couple to yeast G␣- they do to other groups of heptahelical integral membrane proteins [62] it is possible that the endogenous G␣-proteins proteins (see Supplemental Fig. 1B). Nevertheless, some pre- functionally substitute for their human homologues. However, liminary evidence supports this conclusion [18,19,10,11] and we demonstrated that mPR␥ and mPR␣ were also fully capa- steroids 73 (2008) 1160–1173 1171

ble of sensing and responding to progesterone in cells lacking receptor seems to have evolved in vertebrates [63]. Thus, either endogenous G␣-protein. Moreover, we demonstrated the mPRs may represent the original progesterone recep- that constitutive activation of the endogenous G␣-proteins tors. Future experiments to test this hypothesis will involve did not result in activation of the same pathway as PAQR cloning pre-vertebrate Class II PAQRs and testing their ability activation, as would be expected if the mPRs repressed FET3 to respond to progesterone using our assay system. by activating these G␣-proteins. Finally, we demonstrated In summary, we have presented data showing that the that the C-termini of the mPRs, which seem to be essential human mPRs are capable of sensing and responding to proges- for mPR-dependent activation of G␣-proteins in mammalian terone when expressed in yeast cells. Use of this heterologous cells, is not absolutely essential for signal transduction by expression system allowed us to probe the mechanism by mPR␣ and mPR␥ in response to progesterone. These find- which these receptors mediate the effects of progesterone and ings indicate either that the mPRs are not directly coupled present data indicating that these proteins do not absolutely to G-proteins or that these receptors respond to progesterone require heterotrimeric G-proteins to respond to progesterone. by G-protein-dependent and -independent mechanisms. It is We also established a simple system that can be used to begin certainly possible that the C-terminal TM in Class II PAQRs structure/function studies on human mPRs and to explore the allows these receptors to couple directly to G-proteins, but structural features in the agonist steroid that are essential for that this coupling is not the sole mechanism of signal trans- mPR activation. duction. Our assay system has produced additional data that bears Acknowledgements brief discussion. The phylogenetic analysis in Supplemental Fig. 1A suggests that the mPRs are distinct from other PAQR Funding for this study was provided by the National Institutes receptors and form a group of PAQRs that we have called of Health (5 R21 DK074812-02 to TJL) and by the University of Class II. The human genome encodes two other Class II PAQR Florida, Department of Chemistry. proteins called PAQR6 and PAQR9. Figs. 3 and 5 show that proteins in this class of PAQRs are unified by the presence of an eighth TM. We hypothesized that the presence of this Appendix A. Supplementary data eighth TM at the C-terminus of a PAQR is diagnostic of pro- gesterone receptors, even though this additional TM does not Supplementary data associated with this article can be found, seem to be required for progesterone sensing. Not surprisingly, in the online version, at doi:10.1016/j.steroids.2008.05.003. both PAQR6 and PAQR9 sense and respond to progesterone in yeast and have similar steroid specificities to mPR␣,mPR␤ and references mPR␥. PAQR9 is unique in that the EC50 for progesterone acti- vation of this receptor is approximately 10-fold higher than the other four Class II receptors, although it is not yet clear if this is an inherent property of PAQR9 or an artifact of its expres- [1] Kumar R, Thompson EB. The structure of the nuclear sion in yeast. Because of their ability to sense and respond to hormone receptors. Steroids 1999;64(5):310–9. progesterone, we propose the renaming of these receptors to [2] Correia JN, Conner SJ, Kirkman-Brown JC. Non-genomic steroid actions in human spermatozoa. Persistent tickling mPR␦ (PAQR6) and mPR␧ (PAQR9). from a laden environment. 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Semin tree in Supplemental Fig. 1A includes a variety of proteins Reprod Med 2007;25(3):139–53. with the diagnostic eighth TM necessary for their inclusion [6] Bagowski CP, Myers JW, Ferrell Jr JE. The classical in Class II. Intriguingly, the mPR␥ clade of PAQRs, which progesterone receptor associates with p42 MAPK and is includes mPR␦ and mPR␧, actually includes proteins found involved in phosphatidylinositol 3-kinase signaling in in tunicates, lancelets, echinoderms, molluscs, annelids, flat- Xenopus oocytes. J Biol Chem 2001;276(40):37708–14. [7] Boonyaratanakornkit V, Scott MP, Ribon V, Sherman L, worms, cnidarians and even T. adhaerens, an organism at Anderson SM, Maller JL, et al. Progesterone receptor the very base of the metazoan lineage. On the other hand, contains a proline-rich motif that directly interacts with SH3 ␣ ␤ the mPR / clade includes proteins from tunicates, lancelets, domains and activates c-Src family tyrosine kinases. Mol echinoderms, molluscs and annelids. 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H. sapiens PAQR3 CLASS I 240 1000 781 H. sapiens mPRα D. rerio PAQR3 1000 N. vectensis PAQR4 H. sapiens mPRβ 1000 H. sapiens PAQR4 Class II 1000 867 D. rerio PAQR4b H. sapiens mPRγ 886 D. rerio PAQR4a 1000 C. intestinalis PAQR78a 999 H. sapiens PAQR6 1000 C. intestinalis PAQR78b D. rerio PAQR7b H. sapiens PAQR4 890 D. rerio PAQR7a 749 H. sapiens PAQR3 947 G. gallus PAQR7 992 H. sapiens PAQR7 1000 1000 538 S. cerevisiae Izh1p 450 M. domestica PAQR7 α 1000 D. rerio PAQR8 929 S. cerevisiae Izh2p Class I 946 X. tropicalis PAQR8 929 1000 H. sapiens AdipoR1 G. gallus PAQR8 1000 1000 H. sapiens PAQR8 1000 β H. sapiens AdipoR2 962 M. domestica PAQR8 B. floridae PAQR7/8a H. sapiens MMD1 B. floridae PAQR7/8b1 1000 758 993 H. sapiens MMD2 1000 B. floridae PAQR7/8b2 716 B. floridae PAQR7/8b3 998 993 1000 B. floridae PAQR7/8b4 B. cereus Hly3-2 B. floridae PAQR7/8c1 994 406 1000 B. cereus Hly3-1 Class III B. floridae PAQR7/8c2 839 L. gigantea PAQR7/8a Vibro vulnificus Hly3 1000 Capitella sp. PAQR7/8a 1000 537 Capitella sp. PAQR7/8b mPR α -mPR β Clade E. coli yqfa 650 S. purpuratus PAQR78 0.1 L. gigantea PAQR7/8c 979 L. gigantea PAQR7/8b 852 931 L. gigantea PAQR7/8d C. intestinalis PAQR5/6/9/b 612 C. intestinalis PAQR5/6/9/a 1000 C. intestinalis PAQR5/6/9/c C Capitella sp. PAQR5/6 Cellular Organisms CLASS II L. gigantea PAQR5/6 V Escherichia coli (Gram - Eubacteria) Bacillus cereus (Gram + Eubacteria) 871 B. floridae PAQR5/6 ibrio vulnificus (Gram - Eubacteria) Eukartyota Entamoeba histolytica (Protist) 738 H. sapiens PAQR5 γ Fungi/metazoa 1000 Saccharomyces cerevisiae (Fungus) M. domestica PAQR5 Metazoa

559 T

893 G. gallus PAQR5 richoplax adhaerens (Placozoan) 642 Eumetazoa X. tropicalis PAQR5 Nematostella vectensis (Cnidarian) Bilateria 580 D. rerio PAQR5a Schistosoma japonicum (Flatworm) 999 Coelomata 839 999 Capitella sp. (Annelid) Lottia gigantea (Mollusc) 1000 D. rerio PAQR5b1 and 5b2 Deuterostoma Strongylocentrotus purpuratus (Echinoderm) D. rerio PAQR6 Chordata 683 1000 Ciona intestinalis (T Branchiostoma floridae (Lancelet) X. tropicalis PAQR6 Vertebrata

738 H. sapiens PAQR6 Danio rerio (Fish) Xenopus tropicalis (Amphibian) Gallus gallus (Bird) Monodelphis domestica (Marsupial) Homo sapiens (Mammal) 353 1000 M. domestica PAQR6 S. purpuratus PAQR5/6c 957 S. purpuratus PAQR5/6a 1000 S. purpuratus PAQR5/6b S. japonicum PAQR5/6/9 453 T. adhaerens PAQR5/6/9a 938 595 N. vectensis PAQR5/6/9e N. vectensis PAQR5/6/9b unicate) 728 mPR γ Clade N. vectensis PAQR5/6/9d 741 N. vectensis PAQR5/6/9a 445 779 482 N. vectensis PAQR5/6/9c T. adhaerens PAQR5/6/9b 365 T. adhaerens PAQR5/6/9c D. rerio PAQR9 942 X. tropicalis PAQR9 1000 G. gallus PAQR9 335 996 M. domestica PAQR9 1000 H. sapiens PAQR9 Capitella sp. PAQR5/6/9 0.1

Supplemental Fig. 1. Phylogenetic analysis of the PAQR family. For each tree, the length of the tree branches is proportional to the calculated distance between sequences with the scale bar indicating 0.1 substitutions per site. Numbers at the nodes are confidence values that refer to the number times per 1000 trees drawn a particular grouping is made. (A) A bootstrapped phylogenetic tree showing the relationship between PAQR receptors from a variety of eukaryotic and prokaryotic sources. Lines delineate the three Classes of PAQR and grey shading indicates the distinct clades within Class II. The tree is rooted using the sequences in Class III as an outgroup. (B) A bootstrapped phylogenetic tree containing human, yeast and bacterial PAQRs as well as several sequences belonging to two other groups of proteins with a characteristic seven TM structure. One group includes five G-protein-coupled receptor (GPCR) sequences and the other group includes five proteins in the alkaline ceramidase (AlkCer) family of enzymes. The tree is rooted with the clade containing the GPCRs. (C) Taxonomic grouping of the organisms from which the sequences in these trees were derived.